The embodiments of this invention relate generally to systems for preparing viscous materials, such as lipidic mesophases.
Three dimensional protein structures have extremely high commercial value since they allow for the use of rational (structure-based) design and engineering of novel drug molecules that bind to the protein of interest. Furthermore, they facilitate the rational engineering of novel proteins with desired properties. One method of protein X-ray crystallographic structure determination involves: (1) preparation of purified protein; (2) crystallization of the protein; (3) isolation and alignment of single protein crystals in front of an intense and focused X-ray beam; (4) collection of complete X-ray diffraction data sets by rotating the single crystal within the X-ray beam; (5) capturing the diffraction spots on a recording device that measures X-ray spot position and intensity; (6) computational analysis of the X-ray diffraction data to derive experimental electron density maps of the crystal. These maps are in turn used to derive a three dimensional chemical model of the protein that formed the crystal. However, a general problem in the use of X-ray diffraction methods to determine the three-dimensional structures of proteins at near atomic resolution is the rate-limiting step of protein crystallization.
Membrane proteins are a broad class of proteins which bind to and/or traverse a lipid bilayer (membrane) that surrounds all living cells. Membrane proteins are typically involved in the controlled movement of substances and/or signals across the cell membrane. In so doing, membrane proteins enable rapid communication between the inside and outside of living cells. Examples of membrane proteins include ion channels, signaling receptors, hormone receptors, light receptors, and adhesion proteins. Such membrane proteins are the targets of several blockbuster drugs on the market as well as a variety of drugs under development at pharmaceutical companies to treat numerous aliments.
Historically, membrane proteins have been notoriously difficult to crystallize. This is due to their hydrophobic (water hating) and/or lipophilic (fat loving) nature which makes them difficult to purify in large quantity and reduces their overall solubility in aqueous solutions. These factors make it difficult to crystallize membrane protein since they tend to be unstable at concentration in aqueous solutions that are required for the nucleation of crystal growth by crystallization methods used for soluble (non-membrane bound) proteins.
In 1996, Landau and Rosenbusch described the novel use of Lipidic Cubic Phases for the crystallization of membrane proteins. According to this method, detergent solubilized membrane protein is mixed with monoolein (or monopalmitolein) and water (or buffered solutions), followed by multiple rounds of centrifugation. This extensive method allowed for gentle mixing of the materials over 2 to 3 hours to create a viscous, bicontinuous cubic phase, a cured lipid bilayer, extending in three dimensions and permeated by aqueous channels. The membrane proteins can partition into the lipid bilayer and can diffuse in three dimensions which allows them to explore many potential spatial packing configurations that can lead to crystal growth of the protein within lipidic mesophases, such as the so called xe2x80x9cLipidic Cubic Phasexe2x80x9d (LCP).
The Landau and Rosenbusch original LCP crystallization method involves the use of small glass vials into which monoolein, protein and buffered water are added, followed by multiple centrifugations to create the LCP. After the LCP is created, small quantities of dry salt are added and the vials are sealed and incubated. Crystal growth is monitored by examining each glass vial under a stereo microscope. This original lipidic mesophase protocol is tedious, time consuming, and requires more initial protein material than the amount that is necessary for conventional crystallization based on vapor diffusion. The addition of dry salt is time consuming, in particular, as it requires a precision weighing step. In addition, the observation of crystal growth is tedious since it involves multiple tube handling events. Because of these limitations the Landau and Rosenbusch LCP method has generally not been put to use by the protein crystallography community.
In one embodiment of the invention, a coupling device is provided comprising a first receptacle that is operable for coupling with a first syringe; a second receptacle operable for coupling with a second syringe and a channel disposed between the first receptacle and the second receptacle so as to allow fluid to flow from the first receptacle to the second receptacle. The first receptacle can be of a different size from the second receptacle so as to allow different sizes of syringes to be coupled to one another. Such a configuration can be useful as it can facilitate the coupling and the transfer of fluid from a large syringe to a smaller syringe. Furthermore, a tube, such as a needle, can be disposed in the channel so as to facilitate the flow of fluid from one syringe to the other syringe. Also, this embodiment of the invention can be comprised of a heat insulating material, such as PEEK (polyether ether ketone)material, so as to reduce the exchange of heat from a lab worker to the material disposed within the coupling system. Also, a first ferrule can be disposed in the first receptacle so as to facilitate the coupling between the first receptacle and the first syringe. Similarly, a second ferrule can be utilized with the second receptacle to facilitate coupling with the second syringe.
In another embodiment of the invention a method of transferring viscous material, such as lipidic cubic phase material, can be used to transfer the viscous material from a first syringe barrel to a second syringe barrel. This can be accomplished by providing a first syringe barrel containing a volume of viscous material, the first syringe barrel having a first volume size; providing a coupling device; coupling the first syringe barrel with the coupling device; providing yet another syringe barrel having a different volume size from that of the first syringe barrel; coupling this second syringe barrel with the coupling device; and utilizing air pressure to transfer at least a portion of the viscous material to the second syringe barrel from the first syringe barrel. This can facilitate the transfer of fluid or viscous material from a larger syringe to a syringe that is better suited for dispensing the material in small quantities or containers. For example, it can particularly be used for transferring lipidic mesophases, such as LCP, after the lipidic mesophase is mixed by two large syringes (as it is very difficult to mix lipidic mesophases in small syringes). A channel of the coupling device can be used to transfer the viscous material. Furthermore, a needle disposed in the channel can be selected having a sufficiently short length so as to prevent breakage of the syringes during the transfer process.
In another embodiment of the invention, a syringe can be provided for dispensing viscous material, such as in a microwell. For example, a needle of a syringe can be configured so as to have a length of less than about 20 mm and an outside diameter of the needle of about 0.4 mm to about 0.72 mm as well as an inside diameter of the needle of about 0.10 mm to about 0.16 mm. Furthermore, the needle can be sized appropriately so as to dispense lipidic mesophase material without causing breakage of the syringe apparatus during operation.
In yet another embodiment of the invention, a kit of equipment for dispensing or mixing lipidic mesophases or other viscous materials can be provided. For example, a kit can be provided to include: a first syringe having an opening sufficient for receiving lipid material; a second syringe or vessel operable for holding protein solution; and, a coupling device operable for coupling the two syringes together during mixing of the lipid material with the protein solution. Similarly, a smaller syringe can be provided as part of the kit which is operable for dispensing the lipidic mesophase material once it has been mixed. In addition, a coupling device which facilitates the coupling of the large syringe with the smaller syringe as well as the transfer of lipidic mesophase material from the large syringe to the small syringe can be provided. Also, a semi-automatic dispenser can be provided for use with the dispensing syringe and a microwell can be provided for holding mixtures of solution and lipidic mesophase material. The various components of the kit can be provided in a variety of combinations.